Antimicrobial peptides protect coho salmon from Vibrio anguillarum infections

Fish losses from infectious diseases are a significant problem in aquaculture worldwide. Therefore, we investigated the ability of cationic antimicrobial peptides to protect against infection caused by the fish pathogen Vibrio anguillarum. To identify effective peptides for fish, the MICs of certain antimicrobial peptides against fish pathogens were determined in vitro. Two of the most effective antimicrobial peptides, CEME, a cecropin-melittin hybrid peptide, and pleurocidin amide, a C-terminally amidated form of the natural flounder peptide, were selected for in vivo studies. A single intraperitoneal injection of CEME did not affect mortality rates in juvenile coho salmon infected with V. anguillarum, the causative agent of vibriosis. Therefore, the peptides were delivered continuously using miniosmotic pumps placed in the peritoneal cavity. Twelve days after pump implantation, the fish received intraperitoneal injections of V. anguillarum at a dose that would kill 50 to 90% of the population. Fish receiving 200 microg of CEME per day survived longer and had significantly lower accumulated mortalities (13%) than the control groups (50 to 58%). Fish receiving pleurocidin amide at 250 microg per day also survived longer and had significantly lower accumulated mortalities (5%) than the control groups (67 to 75%). This clearly shows the potential for antimicrobial peptides to protect fish against infections and indicates that the strategy of overexpressing the peptides in transgenic fish may provide a method of decreasing bacterial disease problems.     

Strong Synergy between a Eukaryotic Antimicrobial Peptide and Bacteriocins from Lactic Acid Bacteria

The antimicrobial effect obtained upon combining the prokaryotic antimicrobial peptides (AMPs; more commonly referred to as bacteriocins) pediocin PA-1, sakacin P, and curvacin A (all produced by lactic acid bacteria [LAB]) with the eukaryotic AMP pleurocidin (from fish) has been investigated. The three LAB AMPs alone were active against gram-positive Listeria ivanovii bacteria at nanomolar concentrations, whereas they were inactive against gram-negative Escherichia coli bacteria. Pleurocidin alone was active against both of these types of bacteria at micromolar concentrations. Little if any synergy between the LAB AMPs and pleurocidin against the gram-positive L. ivanovii strain was obtained. In contrast, the LAB AMPs and pleurocidin acted highly synergistically against the gram-negative E. coli strain. Nanomolar concentrations of LAB AMPs increased the growth inhibitory potency of pleurocidin by about fourfold. When micromolar concentrations of LAB AMPs were combined with 2 μg of pleurocidin/ml, 100% growth inhibition was attained, whereas pleurocidin alone at a concentration of 2 μg/ml gave no growth inhibition. Most noteworthy, when high concentrations (128 μg/ml) of pleurocidin in the absence of LAB AMPs were used over a long period of incubation (1 week), some growth of E. coli was observed, whereas 16 μg of pleurocidin/ml completely abolished growth in the presence of 64 to 128 ng of LAB AMPs/ml over the same period of time. The results clearly demonstrate that combining eukaryotic and prokaryotic AMPs can greatly increase the specific activity and broaden the target-cell range of these peptides.     

Antimicrobial and antibiofilm activity of pleurocidin against cariogenic microorganisms

Dental caries is a common oral bacterial infectious disease of global concern. Prevention and treatment of caries requires control of the dental plaque formed by pathogens such as Streptococcus mutans and Streptococcus sobrinus. Pleurocidin, produced by Pleuronectes americanus, is an antimicrobial peptide that exerts broad-spectrum activity against pathogenic bacteria and fungi. Moreover, pleurocidin shows less hemolysis and is less toxic than other natural peptides. In the present study, we investigated whether pleurocidin is an effective antibiotic peptide against common cariogenic microorganisms and performed a preliminary study of the antimicrobial mechanism. We assayed minimal inhibitory concentration (MIC), minimal bactericide concentration (MBC) and bactericidal kinetics and performed a spot-on-lawn assay. The BioFlux system was used to generate bacterial biofilms under controllable flow. Fluorescence microscopy and confocal laser scanning microscopy (CLSM) were used to analyze and observe biofilms. Scanning electron microscopy was used to observe the bacterial membrane. MIC and MBC results showed that pleurocidin had different antimicrobial activities against the tested oral strains. Although components of saliva could affect antimicrobial activity, pleurocidin dissolved in saliva still showed antimicrobial effects against oral microorganisms. Furthermore, pleurocidin showed a favorable killing effect against BioFlux flow biofilms in vitro. Our findings suggest that pleurocidin has the potential to kill dental biofilms and prevent dental caries.     

Construction and Characterization of an Escherichia coli Strain Genetically Engineered for Ni(II) Bioaccumulation

An Escherichia coli strain that accumulated Ni(II) was constructed by introducing the nixA gene (coding for a nickel transport system) from Helicobacter pylori into JM109 cells that expressed a glutathione S-transferase–pea metallothionein fusion protein. The resulting strain accumulated 15 μmol of Ni(II) per g (dry weight) from a 10 μM Ni(II) solution, four times the level taken up by JM109 cells. Ni(II) accumulation did not require an energy source, was inhibited by only 50% by 0.1 M NaCl, and occurred over the pH range from 3 to 9.     

Insect antimicrobial peptides show potentiating functional interactions against Gram-negative bacteria

Antimicrobial peptides (AMPs) and proteins are important components of innate immunity against pathogens in insects. The production of AMPs is costly owing to resource-based trade-offs, and strategies maximizing the efficacy of AMPs at low concentrations are therefore likely to be advantageous. Here, we show the potentiating functional interaction of co-occurring insect AMPs (the bumblebee linear peptides hymenoptaecin and abaecin) resulting in more potent antimicrobial effects at low concentrations. Abaecin displayed no detectable activity against Escherichia coli when tested alone at concentrations of up to 200 μM, whereas hymenoptaecin affected bacterial cell growth and viability but only at concentrations greater than 2 μM. In combination, as little as 1.25 μM abaecin enhanced the bactericidal effects of hymenoptaecin. To understand these potentiating functional interactions, we investigated their mechanisms of action using atomic force microscopy and fluorescence resonance energy transfer-based quenching assays. Abaecin was found to reduce the minimal inhibitory concentration of hymenoptaecin and to interact with the bacterial chaperone DnaK (an evolutionarily conserved central organizer of the bacterial chaperone network) when the membrane was compromised by hymenoptaecin. These naturally occurring potentiating interactions suggest that combinations of AMPs could be used therapeutically against Gram-negative bacterial pathogens that have acquired resistance to common antibiotics.     

Potential Importance of Fish Predation and Zooplankton Grazing on Natural Populations of Freshwater Bacteria

The rates of ingestion of natural bacterial assemblages by natural populations of zooplankton (>50 μm in size) were measured during a 19-day period in eutrophic Frederiksborg Slotssø, Denmark, as well as in experimental enclosures (containing 5.3 m³ of lake water). The fish and nutrients of the enclosures were manipulated. In enclosures without fish, large increases in ingestion by zooplankton >140 μm in size were found (up to 3 μg of C liter⁻¹ h⁻¹), compared with values less than 0.3 μg of C liter⁻¹ h⁻¹ in the enclosures with fish and in the open lake. Daphnia cucullata and D. galeata dominated the community of zooplankton of >140 μm. Ingestion rates for zooplankton between 50 and 140 μm decreased after a period of about 8 days, in all enclosures and in the lake, to values below 0.1 μg of C liter⁻¹ h⁻¹. On the last 2 sampling days, somewhat higher values were observed in the enclosures with fish present. The >50-μm zooplankton ingested 48 to 51% of the bacterial net secondary production in enclosures without fish, compared to 4% in the enclosures with added fish. Considering the sum of bacterial secondary production plus biomass change, 35 to 41% of the available bacteria were ingested by zooplankton of >50 μm in the enclosures without fish, compared with 4 to 6% in the enclosures with added fish and 21% in the open lake. Fish predation reduced the occurrence of zookplankton sized >50 μm and thus left a large proportion of the available bacteria to zooplankton sized <50 μm. In fact, there were 4.6 × 10³ to 5.0 × 10³ flagellates (4 to 8 μm in size) ml⁻¹ in the enclosures with fish added as well as in the lake, compared with 0.5 × 10² to 2.3 × 10² ml⁻¹ in the enclosures without fish. This link in the food chain was reduced when fish predation on zooplankton was eliminated and a direct route of dissolved organic matter, via the bacteria to the zooplankton, was established.     

Myxinidin, A Novel Antimicrobial Peptide from the Epidermal Mucus of Hagfish, Myxine glutinosa L.

Fish epidermal mucus contains innate immune components that provide a first line of defense against various infectious pathogens. This study reports the bioassay-guided fractionation and characterization of a novel antimicrobial peptide, myxinidin, from the acidic epidermal mucus extract of hagfish (Myxine glutinosa L.). Edman sequencing and mass spectrometry revealed that myxinidin consists of 12 amino acids and has a molecular mass of 1,327.68 Da. Myxinidin showed activity against a broad range of bacteria and yeast pathogens at minimum bactericidal concentration (MBC) ranging from 1.0 to 10.0 μg/mL. Screened pathogens, Salmonella enterica serovar Typhimurium C610, Escherichia coli D31, Aeromonas salmonicida A449, Yersinia ruckeri 96-4, and Listonella anguillarum 02-11 were found to be highly sensitive to myxinidin at the MBC of 1.0–2.5 μg/mL; Staphylococcus epidermis C621 and yeast (Candida albicans C627) had an MBC of 10.0 μg/mL. The antimicrobial activity of myxinidin was found to be two to 16 times more active than a potent fish-derived antimicrobial peptide, pleurocidin (NRC-17), against most of the screened pathogens. The microbicidal activity of myxinidin was retained in the presence of sodium chloride (NaCl) at concentrations up to 0.3 M and had no hemolytic activity against mammalian red blood cells. These results suggest that myxinidin may have potential applications in fish and human therapeutics.     

Occurrence of Beauvericin and Enniatins in Wheat Affected by Fusarium avenaceum Head Blight

We evaluated Fusarium contamination and the levels of hexadepsipeptide mycotoxins in 13 wheat samples affected by head blight in Finland. Fusarium avenaceum was the dominant species (91%) isolated from all samples, but isolates of F. culmorum (4%), F. tricinctum (3%), and F. poae (2%) also were recovered. Beauvericin (0.64 to 3.5 μg/g) was detected in all 13 samples. Enniatin B (trace to 4.8 μg/g) was detected in 12 samples, enniatin B₁ (trace to 1.9 μg/g) was detected in 8 samples, and enniatin A₁ (trace to 6.9 μg/g) was detected in 10 samples. Ten of 13 strains of F. avenaceum and 2 strains of F. poae and F. tricinctum produced beauvericin in culture on rice (trace to 70, 9.4, and 33 μg/g, respectively). All strains also produced enniatins (trace to 2,700 μg/g). This is the first report on the natural cooccurence of beauvericin and enniatins in wheat infected predominantly by F. avenaceum.     

Casein Fermentate of Lactobacillus animalis DPC6134 Contains a Range of Novel Propeptide Angiotensin-Converting Enzyme Inhibitors

This work evaluated the angiotensin-converting-enzyme (ACE)-inhibitory activities of a bovine sodium caseinate fermentate generated using the proteolytic capabilities of the porcine small intestinal isolate Lactobacillus animalis DPC6134 (NCIMB deposit 41355). The crude 10-kDa L. animalis DPC6134 fermentate exhibited ACE-inhibitory activity of 85.51% (±15%) and had a 50% inhibitory concentration (IC₅₀) of 0.8 mg protein/ml compared to captopril, which had an IC₅₀ value of 0.005 mg/ml. Fractionation of the crude L. animalis DPC6134 fermentate by membrane filtration and reversed-phase high-performance liquid chromatography (HPLC) generated three bioactive fractions from a total of 72 fractions. Fractions 10, 19, and 43 displayed ACE-inhibitory activity percentages of 67.53 (±15), 83.71 (±19), and 42.36 (±11), respectively, where ACE inhibition was determined with 80 μl of the fractions with protein concentrations of 0.5 mg/ml. HPLC and mass spectrometry analysis identified 25 distinct peptide sequences derived from α-, β-, and κ-caseins. In silico predictions, based on the C-terminal tetrapeptide sequences, suggested that peptide NIPPLTQTPVVVPPFIQ, corresponding to β-casein f(73-89); peptide IGSENSEKTTMP, corresponding to α_s1-casein f(201212); peptide SQSKVLPVPQ, corresponding to β-casein f(166-175); peptide MPFPKYPVEP, corresponding to β-casein f(124133); and peptide EPVLGPVRGPFP, corresponding to β-casein f(210-221), contained ACE-inhibitory activities. These peptides were chosen for chemical synthesis to confirm the ACE-inhibitory activity of the fractions. Chemically synthesized peptides displayed IC₅₀ values in the range of 92 μM to 790 μM. Additionally, a simulated gastrointestinal digestion confirmed that the ACE-inhibitory 10-kDa L. animalis DPC6134 fermentation was resistant to a cocktail of digestive enzymes found in the gastrointestinal tract.     

Antimicrobial activity of pleurocidin is retained in Plc-2, a C-terminal 12-amino acid fragment

An analysis of a series of five peptides composed of various portions of the pleurocidin (Plc) sequence identified a l2-amino acid fragment from the C-terminus of Plc, designated Plc-2, as the smallest fragment that retained a antimicrobial activity comparable to that of the parent compound. MIC tests in vitro with low-ionic-strength medium showed that Plc-2 has potent activity against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus but not against Enterococcus faecalis. The antifungal activity of the synthetic peptides against phytopathogenic fungi, such as Fusarium oxysporum, Colletotrichum sp., Aspergillus niger and Alternaria sp., also identified Plc-2 as a biologically active peptide. Microscopy studies of fluorescently stained fungi treated with Plc-2 demonstrated that cytoplasmic and nuclear membranes were compromised in all strains of phytopathogenic fungi tested. Together, these results identify Plc-2 as a potential antimicrobial agent with similar properties to its parent compound, pleurocidin. In addition, it demonstrated that the KHVGKAALTHYL residues are critical for the antimicrobial activity described for pleurocidin.     

Strong synergy between a eukaryotic antimicrobial peptide and bacteriocins from lactic acid bacteria

The antimicrobial effect obtained upon combining the prokaryotic antimicrobial peptides (AMPs; more commonly referred to as bacteriocins) pediocin PA-1, sakacin P, and curvacin A (all produced by lactic acid bacteria [LAB]) with the eukaryotic AMP pleurocidin (from fish) has been investigated. The three LAB AMPs alone were active against gram-positive Listeria ivanovii bacteria at nanomolar concentrations, whereas they were inactive against gram-negative Escherichia coli bacteria. Pleurocidin alone was active against both of these types of bacteria at micromolar concentrations. Little if any synergy between the LAB AMPs and pleurocidin against the gram-positive L. ivanovii strain was obtained. In contrast, the LAB AMPs and pleurocidin acted highly synergistically against the gram-negative E. coli strain. Nanomolar concentrations of LAB AMPs increased the growth inhibitory potency of pleurocidin by about fourfold. When micromolar concentrations of LAB AMPs were combined with 2 micro g of pleurocidin/ml, 100% growth inhibition was attained, whereas pleurocidin alone at a concentration of 2 micro g/ml gave no growth inhibition. Most noteworthy, when high concentrations (128 micro g/ml) of pleurocidin in the absence of LAB AMPs were used over a long period of incubation (1 week), some growth of E. coli was observed, whereas 16 micro g of pleurocidin/ml completely abolished growth in the presence of 64 to 128 ng of LAB AMPs/ml over the same period of time. The results clearly demonstrate that combining eukaryotic and prokaryotic AMPs can greatly increase the specific activity and broaden the target-cell range of these peptides.     

The First Salamander Defensin Antimicrobial Peptide

Antimicrobial peptides have been widely identified from amphibian skins except salamanders. A novel antimicrobial peptide (CFBD) was isolated and characterized from skin secretions of the salamander, Cynops fudingensis. The cDNA encoding CFBD precursor was cloned from the skin cDNA library of C. fudingensis. The precursor was composed of three domains: signal peptide of 17 residues, mature peptide of 41 residues and intervening propeptide of 3 residues. There are six cysteines in the sequence of mature CFBD peptide, which possibly form three disulfide-bridges. CFBD showed antimicrobial activities against Staphylococcus aureus, Bacillus subtilis, Candida albicans and Escherichia coli. This peptide could be classified into family of β-defensin based on its seqeuence similarity with β-defensins from other vertebrates. Evolution analysis indicated that CFBD was close to fish β-defensin. As far as we know, CFBD is the first β-defensin antimicrobial peptide from salamanders.     

Staphylococcus epidermidis Antimicrobial δ-Toxin (Phenol-Soluble Modulin-γ) Cooperates with Host Antimicrobial Peptides to Kill Group A Streptococcus

Antimicrobial peptides play an important role in host defense against pathogens. Recently, phenol-soluble modulins (PSMs) from Staphylococcus epidermidis (S. epidermidis) were shown to interact with lipid membranes, form complexes, and exert antimicrobial activity. Based on the abundance and innocuity of the cutaneous resident S. epidermidis, we hypothesized that their PSMs contribute to host defense. Here we show that S. epidermidis δ-toxin (PSMγ) is normally present in the epidermis and sparsely in the dermis of human skin using immunohistochemistry. Synthetic δ-toxin interacted with neutrophil extracellular traps (NETs) and colocalized with cathelicidin while also inducing NET formation in human neutrophils. In antimicrobial assays against Group A Streptococcus (GAS), δ-toxin cooperated with CRAMP, hBD2, and hBD3. In whole blood, addition of δ-toxin exerted a bacteriostatic effect on GAS, and in NETs, δ-toxin increased their killing capacity against this pathogen. Coimmunoprecipitation and tryptophan spectroscopy demonstrated direct binding of δ-toxin to host antimicrobial peptides LL-37, CRAMP, hBD2, and hBD3. Finally, in a mouse wound model, GAS survival was reduced (along with Mip-2 cytokine levels) when the wounds were pretreated with δ-toxin. Thus, these data suggest that S. epidermidis–derived δ-toxin cooperates with the host-derived antimicrobial peptides in the innate immune system to reduce survival of an important human bacterial pathogen.     

Prediction of Antimicrobial Activity of Synthetic Peptides by a Decision Tree Model

Antimicrobial resistance is a persistent problem in the public health sphere. However, recent attempts to find effective substitutes to combat infections have been directed at identifying natural antimicrobial peptides in order to circumvent resistance to commercial antibiotics. This study describes the development of synthetic peptides with antimicrobial activity, created in silico by site-directed mutation modeling using wild-type peptides as scaffolds for these mutations. Fragments of antimicrobial peptides were used for modeling with molecular modeling computational tools. To analyze these peptides, a decision tree model, which indicated the action range of peptides on the types of microorganisms on which they can exercise biological activity, was created. The decision tree model was processed using physicochemistry properties from known antimicrobial peptides available at the Antimicrobial Peptide Database (APD). The two most promising peptides were synthesized, and antimicrobial assays showed inhibitory activity against Gram-positive and Gram-negative bacteria. Colossomin C and colossomin D were the most inhibitory peptides at 5 μg/ml against Staphylococcus aureus and Escherichia coli. The methods described in this work and the results obtained are useful for the identification and development of new compounds with antimicrobial activity through the use of computational tools.     

Antifungal Activity and Action Mechanism of Histatin 5-Halocidin Hybrid Peptides against Candida ssp

The candidacidal activity of histatin 5 is initiated through cell wall binding, followed by translocation and intracellular targeting, while the halocidin peptide exerts its activity by attacking the Candida cell membrane. To improve antimicrobial activities and to understand the killing mechanism of two peptides, six hybrid peptides were designed by conjugating histatin 5 and halocidin. A comparative approach was established to study the activity, salt tolerance, cell wall glucan binding assay, cytotoxicity, generation of ROS and killing kinetics. CD spectrometry was conducted to evaluate secondary structures of these hybrid peptides. Furthermore the cellular localization of hybrid peptides was investigated by confocal fluorescence microscopy. Of the six hybrid congeners, di-PH2, di-WP2 and HHP1 had stronger activities than other hybrid peptides against all tested Candida strains. The MIC values of these peptides were 1–2, 2–4 and 2–4 μg/ml, respectively. Moreover, none of the hybrid peptides was cytotoxic in the hemolytic assay and cell-based cytotoxicity assay. Confocal laser microscopy showed that di-PH2 and HHP1 were translocated into cytoplasm whereas di-WP2 was accumulated on surface of C. albicans to exert their candidacidal activity. All translocated peptides (Hst 5, P113, di-PH2) were capable of generating intracellular ROS except HHP1. Additionally, the KFH residues at C-terminal end of these peptides were assumed for core sequence for active translocation.     

Salinity Promotes Accumulation of 3-Dimethylsulfoniopropionate and Its Precursor S-Methylmethionine in Chloroplasts

Wollastonia biflora (L.) DC. plants accumulate the osmoprotectant 3-dimethylsulfoniopropionate (DMSP), particularly when salinized. DMSP is known to be synthesized in the chloroplast from S-methylmethionine (SMM) imported from the cytosol, but the sizes of the chloroplastic and extrachloroplastic pools of these compounds are unknown. We therefore determined DMSP and SMM in mesophyll protoplasts and chloroplasts. Salinization with 30% (v/v) artificial seawater increased protoplast DMSP levels from 4.6 to 6.0 μmol mg⁻¹ chlorophyll (Chl), and chloroplast levels from 0.9 to 1.9 μmol mg⁻¹ Chl. The latter are minimum values because intact chloroplasts leaked DMSP during isolation. Correcting for this leakage, it was estimated that in vivo about one-half of the DMSP is chloroplastic and that stromal DMSP concentrations in control and salinized plants are about 60 and 130 mm, respectively. Such concentrations would contribute significantly to chloroplast osmoregulation and could protect photosynthetic processes from stress injury. SMM levels were measured using a novel mass-spectrometric method. About 40% of the SMM was located in the chloroplast in unsalinized W. biflora plants, as was about 80% in salinized plants; the chloroplastic pool in both cases was approximately 0.1 μmol mg⁻¹ Chl. In contrast, ≥85% of the SMM was extrachloroplastic in pea (Pisum sativum L.) and spinach (Spinacia oleracea L.), which lack DMSP. DMSP synthesis may be associated with enhanced accumulation of SMM in the chloroplast.     

Accumulation of Trehalose and Sucrose in Cyanobacteria Exposed to Matric Water Stress

The drought-resistant cyanobacteria Phormidium autumnale, strain LPP₄, and a Chroococcidiopsis sp. accumulated trehalose, sucrose, and both trehalose and sucrose, respectively, in response to matric water stress. Accumulated sugar concentrations reached values of up to 6.2 μg of trehalose per μg of chlorophyll in P. autumnale, 6.9 μg of sucrose per μg of chlorophyll in LPP₄, and 4.1 μg of sucrose and 3.2 μg of trehalose per μg of chlorophyll in the Chroococcidiopsis sp. The same sugars were accumulated by these cyanobacteria in similar concentrations under osmotic water stress. Cyanobacteria that did not show drought resistance (Plectonema boryanum and Synechococcus strain PCC 7942) did not accumulate significant amounts of sugars when matric water stress was applied.     

Purification and Characterization of an X-Prolyl-Dipeptidyl Peptidase from Lactobacillus sakei

An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55°C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu²⁺, Hg²⁺, and Zn²⁺). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. K_m s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 μM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 μM, respectively. Among peptides, β-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed.     

Functional Substitution by TAT-Utrophin in Dystrophin-Deficient Mice

Background

The loss of dystrophin compromises muscle cell membrane stability and causes Duchenne muscular dystrophy and/or various forms of cardiomyopathy. Increased expression of the dystrophin homolog utrophin by gene delivery or pharmacologic up-regulation has been demonstrated to restore membrane integrity and improve the phenotype in the dystrophin-deficient mdx mouse. However, the lack of a viable therapy in humans predicates the need to explore alternative methods to combat dystrophin deficiency. We investigated whether systemic administration of recombinant full-length utrophin (Utr) or ΔR4-21 “micro” utrophin (μUtr) protein modified with the cell-penetrating TAT protein transduction domain could attenuate the phenotype of mdx mice.

Methods and Findings

Recombinant TAT-Utr and TAT-μUtr proteins were expressed using the baculovirus system and purified using FLAG-affinity chromatography. Age-matched mdx mice received six twice-weekly intraperitoneal injections of either recombinant protein or PBS. Three days after the final injection, mice were analyzed for several phenotypic parameters of dystrophin deficiency. Injected TAT-μUtr transduced all tissues examined, integrated with members of the dystrophin complex, reduced serum levels of creatine kinase (11,290±920 U versus 5,950±1,120 U; PBS versus TAT), the prevalence of muscle degeneration/regeneration (54%±5% versus 37%±4% of centrally nucleated fibers; PBS versus TAT), the susceptibility to eccentric contraction-induced force drop (72%±5% versus 40%±8% drop; PBS versus TAT), and increased specific force production (9.7±1.1 N/cm² versus 12.8±0.9 N/cm²; PBS versus TAT).

Conclusions

These results are, to our knowledge, the first to establish the efficacy and feasibility of TAT-utrophin-based constructs as a novel direct protein-replacement therapy for the treatment of skeletal and cardiac muscle diseases caused by loss of dystrophin.     

High-Level Production of Recombinant Human Parathyroid Hormone 1-34

Expression of the synthetic human parathyroid hormone 1-34 [hPTH(1-34)] gene by a gene fusion strategy was demonstrated. hPTH(1-34) was produced at the C terminus of the partner peptides involving amino acids 1 to 97, 1 to 117, or 1 to 139 of a modified Escherichia coli β-galactosidase by linker peptides containing oligohistidine of different lengths. The fusion proteins in the inclusion bodies were rendered soluble with urea and subjected to site-specific cleavage with the secretory type yeast Kex2 protease. Optimal expression and enzymatic processing were achieved in the fusion protein βG-117S4HPT, constructed from amino acids 1 to 117 of β-galactosidase and the linker of HHHHPGGSVKKR. The fusion protein accumulated more than 20% of the E. coli total protein. The hPTH(1-34) was purified up to 99.5% with a good yield of 0.5 g/liter of culture. The purified product was identified as intact hPTH(1-34) by amino acid analysis and N-terminal sequencing.